ASH1L-MRG15 methyltransferase deposits H3K4me3 and FACT for damage verification in nucleotide excision repair

To recognize DNA adducts, nucleotide excision repair (NER) deploys the XPC sensor, which detects damage-induced helical distortions, followed by engagement of TFIIH for lesion verification. Accessory players ensure that this factor handover takes place in chromatin where DNA is tightly wrapped around histones. Here, we describe how the histone methyltransferase ASH1L, once activated by MRG15, helps XPC and TFIIH to navigate through chromatin and induce global-genome NER hotspots. Upon UV irradiation, ASH1L adds H3K4me3 all over the genome (except in active gene promoters), thus priming chromatin for XPC relocations from native to damaged DNA. The ASH1L-MRG15 complex further recruits the histone chaperone FACT to DNA lesions. In the absence of ASH1L, MRG15 or FACT, XPC is misplaced and persists on damaged DNA without being able to deliver the lesions to TFIIH. We conclude that ASH1L-MRG15 makes damage verifiable by the NER machinery through the sequential deposition of H3K4me3 and FACT.

. Gene deletions in U2OS cells. The CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) system was employed for ASH1L, XPC, XPA and XPF gene deletions in human U2OS cells. a List of guide RNA sequences employed for gene deletions. b Scheme illustrating how the human ASH1L gene was targeted for disruption leading to two distinct deletions in exon 2 and exons 11/12. PAM, protospacer adjacent motif; sgRNA, single guide RNA. The ASH1L deletion reduces cell survival and perturbs the cell cycle upon UV damage, consistent with impaired UV lesion excision. a Wild-type (WT), ASH1L -/-, XPC -/and XPA -/cells were exposed to different UV doses and left to recover for 7 days in 24-well plates. b Quantification of colony survival. Colony numbers are expressed as the percentage of unirradiated controls. Mean values ± SD, n = 3 independent experiment, each with 6 technical replicates. c WT and ASH1L -/cells were UV-treated and released from mimosine-induced G1 arrest 6 or 18 h before flow cytometry. DNA synthesis was monitored by EdU incorporation. d Proportion of U2OS cells in the indicated cell cycle phases 6 h after UV irradiation. e Proportion of U2OS cells in the indicated cell cycle phases 18 h after UV irradiation. This analysis revealed that, in response to a UV challenge, the ASH1L deletion decreases the population of S-phase cells, compensated by more cells arrested in G2-M compared to WT controls. Panels d and e show mean values ± SD (n = 3, each experiment with 3 technical replicates). Significance was tested by two-way ANOVA.  The centers of methylation peaks were compiled into a DNA segment ("bin") spanning 64 bp. These center bins were expanded out by increments of 64 bp, thus covering up to ± 2 kilobase pairs away from the peak centers. Sequencing reads derived from CTD-ChIP-seq, XPC-ChIP-seq or HSdamage-seq were mapped to each bin of this region of ± 2 kilobases. Similarly, CPDs and CPD excision rates (during the first 3 h after the UV pulse) were mapped around the center of histone methylation peaks. Lines were finally plotted using the locally weighted scatterplot smoothing method with a default span of 0.75; shaded areas around each line represent 95%  confidence intervals. e ChIP-seq tracks obtained from unirradiated WT and XPC -/cells using anti-XPC antibodies. The profiles span 21 kilobase pairs of chromosome 5. f Baseline XPC occupancy obtained by four independent ChIP-seq analyses of unchallenged WT cells (same color code as in a). Boxplots show medians (normalized to the XPC signal in active promoters), first and third quartiles. Whiskers extend from minimum to maximum values (one-way ANOVA). g Percentage changes of XPC occupancy in different genomic features of WT (panel on the left) and ASH1L -/cells (panel on the right), 1 and 3 h after UV irradiation (mean values of three independent experiments). Thick black line, changes of XPC occupancy at sites of novel ASH1L-deposited H3K4me3 peaks; dotted lines, changes of XPC occupancy in the different genomic features (same color code as in a). h Baseline CPD frequency after UV irradiation of WT and ASH1L -/cells, at the global-genome level and in the different genomic features (same color code as in a; n = 1 HS damage-seq experiment). Boxplots show medians (normalized to the global CPD frequency in WT cells), first and third quartiles. i Normalized CPD excision over 3 h after UV irradiation in the indicated genomic features (n = 1 HS damage-seq experiment). Black, WT cells; grey, ASH1L -/cells. The data of Fig. 3f were corrected for the varying proportion of DNA in each genomic feature.
Supplementary Fig. 6. H3K36me2 marks occur away from CPD sites and CTD occupancy. a Density of unfiltered H3K36me2 peaks obtained by ChIP-seq analysis on WT cells before or after a 3-h recovery post UV (20 J . m -2 ). Unlike H3K4me3, the H3K36me2 peak density is lower in active promoters than in most other genomic features, and not substantially increased upon the UV challenge (mean values of two independent experiments). b The H3K36me2 tracks were filtered for peak height with the top quartile as the threshold (mean values of two independent experiments). c The ~100,000 genomic sites harboring de novo H3K36me2 peaks deposited during 3 h post UV, and their flanking sequences, were subdivided into 64-bp bins. The mean initial abundance of CPDs is indicated for each of these bins. Panel on the left: genome-wide positional correlation demonstrating that H3K36me2 is added preferentially away from CPD sites. Panel on the right: low CPD excision rates in the center of novel H3K36m2 peaks deposited after UV irradiation. The dip in the center of the H3K36me2 peaks is indicative of poor CPD repair. These plots were generated as outlined in Supplementary Fig. 5d   confidence intervals). d Distribution of CTD occupancy relative to initial CPD formation and CPD excision. The peaks of the CTD of ASH1L detected 3 h after UV were filtered with the 75 th percentile as the threshold. Shown are CPD densities around the center of these CTD peaks (panel on the left) and CPD excision rates during a 3-h recovery, again relative to the center of CTD peaks (panel on the right). Shaded areas around each line represent 95% confidence intervals.
The purified DNA was mixed with 1.5 μl of O3P primer (20 μM) and an equal volume of NEBNext Ultra II Q5 Master Mix (New England Biolabs), then incubated under the following conditions in thermocycler T100 (Bio-Rad Laboratories) for polymerase extension: 50 sec at 98°C, 5 min at 65°C and on hold at 37°C. To digest the excessive amount of primer, 1.5 μL exonuclease I (New England Biolabs, M0293) were added to the extension mixture, followed by incubation at 37°C for 15 min. The mixture was purified with a 0.9x volume of AMPure XP (37 µl, 25 µl MilliQ) and eluted with 20 μl 0.1x TE buffer. The eluate was mixed with 2 μl of SH primer (10 μM), 25 μl 1x B&W buffer [5 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.1% (vol/vol) Tween 20] and subjected to a slow annealing process using the thermocycler under the following conditions: 2 min at 98°C, then cooling at 1 min/°C from 97°C to 76°C, 5 min/°C from 75°C to 55°C, 1 min/°C from 54°C to 25°C, and on hold at 4°C. The annealing product was stored at -20 °C for later processing. Next, 10 μl Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific, 65001) were washed twice with 1x B&W buffer, resuspended with 5 μl 5x binding buffer [50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 2.5 M NaCl, 0.1% (vol/vol) Tween 20, 0.1% (wt/vol) CA-630, 25 mM MgCl2)] and added to the annealing product. The mixture was rotated 1 h at 4°C on the tube revolver in oscillation mode. The supernatant was transferred to a new 1.5-ml tube, the beads were washed with 50 µL 1x B&W buffer, and the supernatants were pooled. The DNA in pooled supernatants was purified by ethanol precipitation by adding 1 μl GlycoBlue and 250 µl of ethanol. The air-dried pellet was resuspended in 6.5 μl 0.1x TE buffer and denatured by heating to 98°C for 2 min, then immediately placed on ice. The denatured DNA was centrifuged at 12,000 g for 30 s to collect all solution at the bottom of tube. Then, 1 μl AD2 (40 μM) and 7.5 µl of Instant Stick Ends Ligase Master Mix (New England Biolabs, M0370) were added. The mixture was kept overnight at 4 °C and purified with 0.8x volume AMPure XP (40 µl, 35 μl Milli-Q), then eluted with 16 μl 0.1x TE buffer. The eluted DNA was amplified using NEB Next Ultra II Q5 Master Mix with indexing primers for Illumina. The amplified products were purified by 0.9x AMPure XP beads and eluted with 25 μl 0.1x TE buffer. The concentration of eluted DNA was determined using Quantus Fluorometer (Promega). An equal amount of each library sample (³ 20 ng) was pooled and purified again by 0.9x AMPure XP beads to remove residual primer-dimers, then eluted using 25 µl 10 mM Tris-HCl buffer (pH 8.0). The mixture was further diluted to 5 ng/µl and sent for sequencing. The pooled libraries were sequenced as 1x100 bp on an Illumina NovaSeq6000 sequencer.

Omni-ATAC-seq
An improved ATAC-seq method 1 was used to generate chromatin accessibility profiles in wildtype and ASH1L -/cells. After UV-C irradiation at 20 J . m -2 , cells were allowed to recover for 3 h at 37°C in fresh medium or were processed directly in the case of unirradiated controls. To digest the DNA of dead or damaged cells before harvesting, cells were treated with deoxyribonuclease I (Sigma-Aldrich, D4513-1VL) at a final concentration of 200 units/mL and resuspended in Hanks' Balanced Salt Solution (Sigma-Aldrich, 55037C) for 30 min at 37°C. Cells were then washed three times with PBS, removed by trypsinization and counted. A cell viability of > 90% was confirmed by the addition of 0.4% (wt/vol) trypan blue solution (Thermo Fisher Scientific, 15-250-061).
Next, 50,000 cells were transferred to a 1.5-mL DNA LoBind tube (Eppendorf). Cells were pelleted at 2,500 g for 5 min at 4°C. The supernatant was removed and the cell pellet was resuspended in 50 µL of ice cold ATAC-seq lysis buffer [10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (wt/vol) NP-40, 0.1% (wt/vol) Tween 20, and 0.01% (wt/vol) digitonin] and left on ice for 3 min before adding 1 mL of ice-cold ATAC-seq wash buffer [10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (wt/vol) Tween 20]. Subsequently, nuclei were pelleted at 2,500 g for 10 min at 4 °C and the supernatant was aspirated. The nuclei were resuspended and exposed to the transposition reaction following the manufacturer's protocol (Diagenode, C01080002). This assay is based on the use of the transposase Tn5, which "tagments" the DNA by cleaving and tagging, through the ligation of sequencing adapters, accessible chromatin.
Transposition reactions were stopped by adding 5 volumes of DNA-binding buffer from the Zymo Concentrator-5 kit (Zymo Research, D4013). DNA was purified using this kit according to the manufacturer's instructions including a 10,000 g centrifugation step. Samples were eluted into DNA LoBind tubes with 23 µL of DNA elution buffer pre-warmed to 50°C. After an incubation of 20 min and a 30-sec centrifugation, the eluate was re-added to the column, incubated for 5 min and centrifuged again for 30 sec.
The transposed DNA fragments were pre-amplified for 5 cycles with the LabCycler (SensoQuest) using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and unique dual indices (primers are listed in Supplementary Table 4). After 5 cycles, samples were removed from the thermocycler and stored on ice. To avoid overamplification, qPCR was used to determine the number of additional cycles to run: 5 µL of the pre-amplified mixture were further amplified on the CFX384 Touch Real-Time PCR detection system (Bio-Rad). The required number of additional amplification cycles corresponded to the maximum relative fluorescence units divided by 4. This additional number of cycles was run using the remaining pre-amplified DNA. Thereafter, a double-sided clean-up with AMPure XP beads (Beckman Coulter, A63880) was performed. All steps were conducted at room temperature and in DNA LoBind tubes. To remove larger fragments (> 1,000 bp), 0.5 volumes of AMPure XP beads were thoroughly mixed with each sample by pipetting up and down 10 times. Samples were incubated for at least 20 min. Tubes were placed on a magnetic rack for 5 min before the supernatant was transferred to a new tube. Smaller fragments (75-100 bp) were removed by adding a 1.3-fold volume of AMPure XP beads, resulting in a final beads-tosample ratio of 1.8. After a 20-min incubation, the tubes were placed on the magnetic rack for another 5 min. The supernatant was removed and the beads remaining in the tube were washed with freshly made 80% ethanol by pipetting ethanol over the beads 10 times. After the last wash step, ethanol was removed and the tubes were left on the magnetic rack for 10 min with the caps open to allow all residual ethanol to evaporate. Beads were resuspended in 20 µL Milli-Q water and again placed on the magnetic rack for 5 min. The supernatant was transferred to a new tube and the procedure was repeated once more before DNA concentration was determined using the Invitrogen Qubit 4 Fluorometer. To verify a desired fragment size of 200-1,000 base pairs, purified libraries were analyzed on the 4200 TapeStation system from Agilent using the High Sensitivity D1000 ScreenTape assay. Next, libraries were pooled at equimolar concentrations and sent to be sequenced to 50 million reads per sample on an Illumina Novaseq 6000 (SP flowcell) as 50-base pair reads.
Raw reads (FASTQ files) were processed based on the ENCODE ATAC-seq pipeline for paired-end reads. First, adapters were detected and trimmed (cutadapt v.1.9.1). Next, the trimmed reads were aligned (bowtie2 v.2.4.5) to the human reference genome build GCA hg38 excluding chromosome Y but including non-canonical contigs. These alignments underwent further filtering steps (samtools v.1.7, bedtools v.2.29.2, Picard v.2.23.8) to exclude reads that were PCR or optical duplicates, were unmapped or non-primary alignments, failed platform and/or vendor quality checks, had mapping quality scores below 30, or mapped to ENCODE blacklisted regions, non-canonical chromosomes or mitochondrial DNA. The filtered alignments were used to call peaks (macs2 v.2.2.7.1 with default parameters), which were used to verify replicability across biological replicates (idr v.2.0.4.2). ATAC-seq signal tracks were generated and uploaded to the IGV 2,3 to visualize continuous signals. Custom bash and R scripts were used to find the distribution of ATACseq peaks under the different conditions.

Mass spectrometry
The liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of trypsintreated peptides was performed on an Q Exactive mass spectrometer (Thermo Scientific) equipped with a Digital PicoView source (New Objective) and coupled to a nanoAcquity UPLC (Waters). Solvent composition at the two channels was 0.1% (vol/vol) formic acid for channel A and 0.1% formic acid, 99.9% (vol/vol) acetonitrile for channel B. Column temperature was 50°C. For each sample, 2 μL of peptides were loaded on a commercial Symmetry C18 trap column (5 µm, 180 µm x 20 mm, Waters) connected to a BEH300 C18 column (1.7 µm, 75 µm x 150 m, Waters Inc.). The peptides were eluted at a flow rate of 300 nL/min with a gradient from 5 to 35% B in 60 min, 35 to 60% B in 5 min and 60 to 95% B in 10 min before equilibrating back to 5% B.
The mass spectrometer was operated in data-dependent mode (DDA). Full-scan MS spectra (350−1500 m/z) were acquired at a resolution of 70,000 at 200 m/z after accumulation to a target value of 3,000,000, followed by higher-energy collision dissociation (HCD) fragmentation on the twelve most intense signals per cycle. Ions were isolated with a 1.2 m/z isolation window and fragmented by HCD using a normalized collision energy of 25%. HCD spectra were acquired at a resolution of 35,000 and a maximum injection time of 120 ms. The automatic gain control (AGC) was set to 100,000 ions. Charge state screening was enabled and singly and unassigned charge states were rejected. Only precursors with intensity above 25,000 were selected for MS/MS. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 40 s, and the exclusion window tolerance was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.1010 and 445.1200. The mass spectrometry proteomics data were handled using the local laboratory information management system (LIMS) 4 and all relevant data have been deposited to the ProteomeXchange consortium via the PRIDE (http;//www.ebi.ac.uk/pride) repository.
The acquired raw MS data were processed by MaxQuant v.2.0.1.0, followed by protein identification using the integrated Andromeda search engine 5 . Spectra were searched against the Uniprot Homo sapiens reference proteome (taxonomy 9606, canonical version from 2019-07-09), concatenated to its reversed decoyed fasta database and common protein contaminants. Methionine oxidation and N-terminal protein acetylation were set as variable. Enzyme specificity was set to trypsin/P allowing a minimal peptide length of 7 amino acids and a maximum of two missed cleavages. MaxQuant Orbitrap default search settings were used with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 10 ppm. The applied cutoffs entailed a minimal score for modified peptides of 40, and a minimal delta score for modified peptides of 6. The minimum number of unique peptides for protein identification was 2 peptides. The protein false discovery rate (FDR) was 1.0% and the peptide FDR was 0.1%.